The Biofilm Exopolysaccharide Polysaccharide Intercellular Adhesin—A Molecular and Biochemical Approach - Bacterial Pathogenesis: Methods and Protocols - page 98

3.3.2. Immuno-Dot Blot Analysis
To quantify PIA production, use equal amounts of bacterial cells and culture
supernatants. The protocol is modified from Gerke et al. (5) .
1. Spot 2-μL sample aliquots to a nitrocellulose membrane, let the membrane dry,
and block with 0.5% skim milk in TBS buffer overnight.
2. Incubate the membrane for at least 2 h with blocked PIA-antiserum (1:5000)
and wash membrane for3×5minwith TBS buffer.
3. Incubate membrane for 2 h with IgG-alkaline-phosphatase conjugate (1:5000).
4. Detect spots by chromogenic reaction with 5 mL 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium premix solution.
5. Quantify immunological reaction of PIA (spot), e.g., by using a scanner and Total
Lab Version 2003 software (Nonlinear USA, Durham, NC) ( see Note 6 ).
3.3.3. Immunofluorescence Microscopy
1. Heat-fix 15 μL of aliquots of bacterial cell suspensions onto glass microscope
slides.
2. Incubate fixed cells with 30 μL of PIA antiserum (diluted 1:50 in TBS) for
30 min at 37 °C in a wet chamber.
3. Wash slides for3×5minwith TBS buffer and let them air-dry.
4. Incubate bound antibodies with 30 μL of fluorescein isothiocyanate-conjugated
anti-rabbit immunoglobulin G antibody, diluted at 1:80 in TBS, for 30 min at
37 °C in a wet chamber.
5. Wash glass slide twice with TBS and double-distilled water for 5 min, air-dry
glass slide.
6. Detect PIA under a fluorescence microscope at a magnification of ×1000.
3.4. Analysis and Detection of PIA by Chromatography
3.4.1. PIA Analysis by Size Exclusion Chromatography/Electro-Spray
Ionization Mass Spectrometry
This method employs SEC coupled to electro-spray ionization mass
spectrometry (ESI/MS) and can be applied to culture filtrate samples and crude
PIA extracts isolated from cell surfaces. The acquisition of ESI mass spectra of
PIA in positive ion mode is based on the ionization of groups such as hydroxyl
groups on sugar residues, or in the case of deacetylated PIA on already ionized
amino groups. On the size exclusion columns used under the specific buffer
conditions, PIA elutes earlier and separate from most other molecules, soon
after the exclusion volume.
1. Purify PIA as described in Subheading 3.2 .
2. Perform SEC/ESI-MS at a flow rate of 0.5 or 1 mL/min using 0.2% acetic acid.
An Agilent 1100 system coupled to a Trap VL or SL mass spectrometer was used.